Application Note
Purification of soil metagenomic DNA with the MasterPure™ Complete KitIntroduction

Note: It is critical to follow the recommended centrifugal speeds, asStudying the rich diversity of the soil metagenome can prove challenging,indicated in the protocol, for efficient recovery of the microbes from theas most bacteria are unculturable and many substances present in soil aresoil samples.

inhibitory to downstream applications. The MasterPure™ Complete DNAand RNA Purification Kit can be easily adapted to purify metagenomic1.

conical tube, then add 10 mL of PBS with 0.1% Tween 20.

DNA from microoganisms present in soil. The isolated, randomly sheared
DNA is high molecular weight (~40 kB), free of humic and fulvic acids,
and suitable for downstream applications including next gen sequencing,

2.

3.

sequencing and PCR applications, we recommend using the NxSeq®

4.

Add 20 mL of the remaining PBS with 0.1% Tween 20 (per extraction)
to the original soil pellet, and mix by vortexing at maximum speed for

UltraLow DNA Library Kit and FailSafe™ PCR Systems, respectively.

Methods

Centrifuge the soil suspension at 1,600 × g for 4 minutes in a tabletop
centrifuge. Pour the supernatant into a new 50 mL tube.

DNA in conjunction with the CopyControl™ Fosmid Library Production
Kit or CopyControl HTP Fosmid Library Production Kit. For next gen

Mix by vortexing at maximum speed for 1 minute to disperse anddissociate the soil particles.

PCR, and fosmid library production. For increased reliability andsimplicity in fosmid library production, we recommend using the isolated

Add 1 g of wet soil (or 200–300 mg of compost) to a 50 mL screw-cap

1 minute.
5.

Centrifuge the soil suspension at 900 × g for 3 minutes in a tabletop

Use the reagents from the MasterPure Complete DNA & RNA Purification

centrifuge. Important! Do not exceed 900 × g. Remove theKit. You will need to supply the following additional reagents:

supernatant and combine it with the previously collected supernatantfrom step 3.

• Phosphate-buffered saline (PBS)• Ready-Lyse™ Lysozyme Solution (Lucigen Cat. No. R1804M)

6.

proceed to step 7.

• 0.45-μm filter membranes (e.g., Sterlitech Cat. No. CA04547100)• 1.2-μm filter membranes (e.g., Sterlitech Cat. No. CA1247100)

Re-extract the soil pellet again by repeating steps 4 and 5, and then7.

Briefly centrifuge the pooled supernatant at 900 × g for 2 minutes ina tabletop centrifuge. Transfer the supernatant to a fresh 50 mL tube.

• Filtration apparatus (Millipore Sigma Cat. No. XX1004700, orequivalent)• Miracloth filtration material (Millipore Sigma) or sterile cheesecloth• 50 mL conical tubes

Filtration, Lysis, and Protein Precipitation
1.

Miracloth or sterile cheesecloth.

• 1.7 mL microcentrifuge tubes• Tween® 20 (molecular biology grade)

2.

3.

Pass the collected filtrate through the 0.45 μm filter membrane using
an appropriate filter apparatus in order to trap the microbial mass on

Extraction

the filter. Retain the filter membrane.

Each extraction will use 50 mL of PBS with 0.1% Tween 20. Before starting,prepare enough buffer for the number of samples you will be purifying byPrefilter the sample through the 1.2 µm filter membrane using anappropriate filter apparatus. Collect the filtrate.

• Isopropanol• 70% ethanol (prepared from absolute ethanol)

Pour the entire collected supernatant (50 mL) through four layers of4.

Using forceps and scissors presoaked in 70% ethanol, remove theadding 50 μL of Tween 20 per 50 mL of PBS to achieve a final concentration

membrane from the filter apparatus, cut the membrane in half, andof 0.1% Tween 20.

place each half (rounded side down) along the side (near the bottom)

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lucigen.com | ph 888 575 9695 | f 608 831 9012 | United States
ISO 13485 Certified. For research or investigational use only.

of a 50 mL sterile conical tube. The upper surface of the filter needs
to face the center (not the wall) of the tube. Do not allow the filter
membrane to dry out.
5.

Prepare the Filter Wash Buffer by adding 1.5 μL of Tween 20 to
1.5 mL of PBS immediately before use. Add 1.5 mL of Filter Wash
Buffer containing 0.1% Tween 20 to the filter pieces in the tube.

6.

Vortex the tube at the low speed setting to rewet the filter pieces,then increase the setting to the highest speed.

7.

18. Pellet the DNA by centrifuging for 10 minutes at 20,000 × g, ormaximum speed, in a microcentrifuge at 4°C.
19. Use a pipet tip to remove the isopropanol without dislodging theDNA pellet. Briefly pulse-centrifuge the sample, and remove any
residual liquid with a pipet tip without disturbing the pellet.
20. Add 500 μL of 70% ethanol without disturbing the pellet. Centrifuge
for 5 minutes at 20,000 × g, or maximum speed, in a microcentrifuge

centrifuge the tube at 14,000 × g or top speed in a microcentrifuge

at 4°C.
21. Use a pipet tip to remove the ethanol without dislodging the DNA

Resuspend the cell pellet in 300 μL of TE Buffer, then add 2 μL ofpellet. Briefly pulse-centrifuge the sample and remove any residual

Ready-Lyse Lysozyme Solution and 1 μL of RNase A to the cellliquid with a pipet tip without disturbing the pellet.

suspension. Mix, and centrifuge briefly.
9.

tube several times.

Transfer the cell suspension to a clean microcentrifuge tube, thenfor 2 minutes to pellet the cells. Discard the supernatant.

8.

17. Add 570 μL of isopropanol to the supernatant. Mix by inverting theIncubate the tube at 37°C for 30 minutes.

10. Add 300 μL of 2X Tissue and Cell Lysis Solution and 1 μL ofProteinase K to the tube. Mix by vortexing.
11. Briefly pulse-centrifuge the tube to ensure that all of the solution is inthe bottom of the tube.
12. Incubate at 65°C for 15 minutes.
13. Place on ice for 3–5 minutes.
14. Add 350 μL of MPC Protein Precipitation Reagent to the tube and mixby vortexing vigorously for 10 seconds.
15. Pellet the debris by centrifuging for 10 minutes at 20,000 × g, ormaximum speed, in a microcentrifuge at 4°C.
16. Transfer the supernatant to a clean 1.7 mL microcentrifuge tube anddiscard the pellet.

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lucigen.com | ph 888 575 9695 | 608 831 9011 | United States
ISO 13485 Certified. For research or investigational use only.

22. Air-dry the pellet for 8 minutes at room temperature.
Note: Do not over-dry the DNA pellet.
23. Resuspend the DNA pellet in 40 μL of TE Buffer.
24. Validate the size and concentration of the isolated DNA byelectrophoresis on a 1% agarose gel.
The isolated DNA is ready for PCR, next gen sequencing, or end-repair
and subsequent cloning into a vector for construction of a fosmid library.
If using the CopyControl Fosmid Library Production Kit, proceed withend-repair (Section B) and ligation (Section E) in the supplied protocol(#MA171E: CopyControl Fosmid Library Production Kits).

NxSeq is a registered trademark, and CopyControl, FailSafe, MasterPure, andReadyLyse are trademarks of Lucigen.
Tween is a registered trademark of Croda International PLC.